There is currently a need in drug discovery and development and in general biological research for methods and apparatus for accurately performing cell-based assays. Cell-based assays are advantageously employed for assessing the biological activity of chemical compounds and the mechanism-of-action of new biological targets. In a cell-based assay, the activity of interest is measured in the presence of both competing and complementary processes. As pertains to chemical compound screening, information is available as to the specific activity of the compound. For example, it is possible to assess not only whether a compound binds the target of the assay, but also whether it is an agonist or an antagonist of the normal activity of the target. Frequently, the target is a cell-surface receptor. In some signaling pathways, the member of the pathway of greatest potential therapeutic value is not the receptor but an intracellular signaling protein associated with the receptor. It is, therefore, desirable to develop methods to assay activity throughout the pathway, preferably in the cellular milieu.
In addition, there is a need to quickly and inexpensively screen large numbers of chemical compounds. This need has arisen in the pharmaceutical industry where it is common to test chemical compounds for activity against a variety of biochemical targets, for example,
receptors, enzymes and nucleic acids. These chemical compounds are collected in large libraries, sometimes exceeding one million distinct compounds. The use of the term chemical compound is intended to be interpreted broadly so as to include, but not be limited to, simple organic and inorganic molecules, proteins, peptides, nucleic acids and oligonucleotides, carbohydrates, lipids, or any chemical structure of biological interest.
In the field of compound screening, cell-based assays are run on collections of cells. The measured response is usually an average over the cell population. For example, a popular instrument used for ion channel assays is disclosed in U.S. Pat. No. 5,355,215. A typical assay consists of measuring the time-dependence of the fluorescence of an ion-sensitive dye, the fluorescence being a measure of the intra-cellular concentration of the ion of interest which changes as a consequence of the addition of a chemical compound. The dye is loaded into the population of cells disposed on the bottom of the well of a multiwell plate at a time prior to the measurement. In general, the response of the cells is heterogeneous in both magnitude and time. This variability may obscure or prevent the observation of biological activity important to compound screening. The heterogeneity may arise from experimental sources, but more importantly, heterogeneity is fundamental in any population of cells. Among others, the origin of the variability may be a consequence of the life-cycle divergence among the population, or the result of the evolutionary divergence of the number of active target molecules. A method that mitigates, compensates for, or even utilizes the variations would enhance the value of cell-based assays in the characterization of the pharmacological activity of chemical compounds.
Quantification of the response of individual cells circumvents the problems posed by the non-uniformity of that response of a population of cells. Consider the case where a minor fraction of the population responds to the stimulus. A device that measures the average response will have less sensitivity than one determining individual cellular response. The latter method generates a statistical characterization of the response profile permitting one to select the subset of active cells. Additional characterization of the population will enhance the interpretation of the response profile.
Various measurement devices have been used in the prior art in an attempt to address this need. Flow-cytometer-based assays are widely practiced and measure cell properties one at a time by passing cells through a focused laser beam. Several disadvantages accompany this method. Most important to the pharmaceutical industry is that assays can not readily be performed on compounds disposed in microtiter plates. In addition, the throughput is poor, typically 10-100 seconds per sample, the observation time of each cell is <1 ms, prohibiting kinetic assays, and finally, only the cell-averaged signal can be determined.
In addition, many assays require determination of the relative locations of the fluorescence signals. Devices called scanning cytometers, as disclosed in U.S. Pat. No. 5,107,422 and U.S. Pat. No. 5,547,849, are widely used for imaging single cells. In order to gain acceptable speed, these devices operate at low (˜5-10 μm) resolution. Thus, these devices offer little advantage over flow cytometers for assays requiring spatial information on the distribution of the fluorescence signals.
An additional alternative technology is the fast-camera, full-field microscope. These devices have the ability to obtain images at a resolution and speed comparable to the present invention, on certain samples. However, they are not confocal and are consequently susceptible to fluorescence background and cannot be used to optically section the sample. In addition, simultaneous, multi-parameter data is not readily obtained. In contrast to the prior art, the present invention can be used to perform multi-parameter fluorescence imaging on single cells and cell populations in a manner that is sufficiently rapid and versatile for use in compound screening. Methods and apparatus are provided for obtaining and analyzing both the primary response of individual cells and additional measures of the heterogeneity of the sample population. In addition, the locations of these multiple fluorophores can be determined with sub-cellular resolution. Finally, the present invention can be used to image rapidly changing events at video-rates. Together these capabilities enable new areas of research into the mechanism-of-action of drug candidates.
The present invention may also be employed in an inventive fluorescence-based biochemical assay, somewhat analogous to the surface scintillation assay (“SSA”) which is among the more widely used methods for screening chemical compounds.
FIGS. 1A-1F depict the steps of a receptor-binding SSA. In FIG. 1A, soluble membranes 10 with chosen receptors 12 are added to a well 20 containing a liquid 30. These membranes are isolated from cells expressing the receptors. In FIG. 1B, radio-labeled ligands 14 are added to the well. The ligand is known to have a high binding affinity for the membrane receptors. The most common radio labels are 3H, 35S, 125I, 33P and 32P. In FIG. 1C, beads 16 are added to the well. The beads are coated with a material, such as wheat germ agglutinin, to which the membranes strongly adhere. The beads have a diameter of 3-8 μm and are made of plastic doped with a scintillant. Alternatively, the order of the operations depicted in FIGS. 1B and 1C may be interchanged.
The radiolabels decay by emitting high energy electrons, or beta particles, which travel approximately 1-100 μm before stopping, depending on the radio-isotope. If the radiolabels are bound to the membranes attached to the beads, the beta particles may travel into the beads and cause bursts of luminescence. If the radio-labels are dispersed throughout the liquid, the emitted beta particles will not generally excite luminescence in the beads. In FIG. 1D, the luminescence of the beads caused by decay of the radio labels is detected. In FIG. 1E, a test compound 18 is added to the well. The purpose of the assay is to determine the extent to which this compound will displace the radio-labeled ligands. If radio-labeled ligands are displaced and diffuse into the liquid, the luminescence of the beads will be reduced. In FIG. 1F, the luminescence of the beads is again detected. By measuring the reduction in luminescence, the activity of the test compound can be determined.
FIGS. 2A-2F depict an alternative embodiment of a receptor-binding SSA. This embodiment is essentially the same as that described in FIGS. 1A-1F except that instead of using beads, the embodiment shown in FIGS. 2A-2F uses a well bottom 22 made of plastic doped with scintillant and coated with a material to which the membranes adhere. Consequently, instead of detecting the luminescence of the beads, the embodiment shown in FIGS. 2A-2F detects the luminescence of the well bottom.
FIGS. 3A-3D depict the steps of an embodiment of an enzyme SSA. In FIG. 3A, scintillant-doped beads 40 with radio-labeled peptides 42 attached thereto are added to a well 50 containing a liquid 60. In FIG. 3B, a test compound 44 is added to the well. In FIG. 3C, enzymes 46 are added to the well. If not inhibited, enzymes 46 will cleave radio-labeled peptides 42 from beads 40. As a result, the radio label will diffuse into the solution, and radio-label decay will not produce luminescence in beads 40. If, on the other hand, test compound 44 inhibits enzymes 46, typically by blocking the enzyme active site, enzymes 46 will not cleave the radio label and the decay of the radio label will produce luminescence in the beads. In FIG. 3D, the luminescence of the beads is measured and the activity of the test compound can be determined.
FIGS. 4A-4D depict an alternative embodiment of an enzyme SSA. In FIG. 4A, radio-labeled peptides 42 are attached to a scintillant-doped well bottom 52. In FIG. 4B, the test compound 44 is added to the well. In FIG. 4C, enzymes 46 are added to the well. In FIG. 4D, the luminescence of the well bottom is measured to determine the activity of the test compound.
The above examples illustrate the general principle of the SSA, namely that the activity of interest is assayed by a change in the number of radio labels within a radio-decay length of the scintillant. One of the attractions of SSAs is that the radio labels not attached to the scintillant need not be removed from the well in a wash step. That is SSAs are homogeneous assays.
A radioimmunoassay (RIA) is a specific form of a receptor binding assay in which the receptor is an antibody and the ligand is most often a natural or synthetic peptide, protein, carbohydrate or small organic molecule. RIAs are an indirect method for measuring the concentration of ligand in any prepared sample, most often a biological sample such as plasma, cerebrospinal fluid, urine, or cellular extract. In a standard RIA, the antibody has a specific affinity for the ligand and the assay contains the antibody, a fixed concentration of radiolabeled ligand and an unknown concentration of non-labelled ligand. The concentration of the unlabelled ligand is determined by the degree to which it binds to the antibody and thereby blocks binding of the labelled ligand. RIAs are most often performed as heterogenous assays that require the separation of bound ligand from unbound ligand with a wash step. RIAs have also been developed using an SSA configuration in which the antibody receptor is attached to a scintillant filled bead and the wash step is eliminated.
SSAs and RIAs, however, suffer from a number of disadvantages. First, these assays require handling radioactive material, which is both expensive and time consuming. Second, these assays are only effective in large wells. The rate of luminescence emission from the beads or well bottoms is proportional to the beta particle emission rate. A typical 3H assay yields less than one detected photon per 3H decay. To increase the speed of the assay, the quantity of radio-labeled ligand must be increased, and correspondingly the quantities of membranes, beads and test compound. In order to perform a tritium SSA in 10-60 seconds, 107 beads must be used. This quantity of beads requires a well of approximately 150 μL. SSAs are not effective in the μL-volume wells desirable for screening large numbers of compounds.
As described below, the present invention, inter alia, replaces the radio-labeled ligands of the SSA and the RIA with fluorescent-labeled ligands. In so doing, it introduces a homogenous format for the RIA and it advantageously retains the homogeneous format of the SSA. This is particularly important in μL-volume wells, for which surface tension renders washing impractical. However, in a homogeneous format, fluorescence can be a problem as can be illustrated with the receptor-binding assay. When the test compound is added, some fluorescent-labeled ligands are displaced and diffuse freely throughout the volume of the well, while others remain attached to the membranes. It is the fluorescence of the fluorescent-labeled ligands attached to the membranes that is used to determine the activity of the test compound. If the fluorescence is detected from the entire well, however, the emission from the fluorescent-labeled ligands in the volume of the well will obscure the emission from the fluorescent-labeled ligands attached to the membranes.
One method addressing this problem is described in U.S. Pat. No. 5,355,215 to Schroeder et al. and shown in FIGS. 5A and 5B. According to the Schroeder et al. method, the samples are illuminated by a beam 134 of light that is directed at the bottom of the well at an oblique angle, shown as A in FIG. 5, so that it does not illuminate the entire well. In addition, while the beam illuminates area 114′, fluorescence is detected only from area 114a which is under the well volume which receives the least amount of illumination.
The Schroeder et al. method, however, suffers from a number of disadvantages. First, because it detects only a small portion of the well bottom, the Schroeder et al. method can only be performed with a sufficient degree of accuracy on fairly large wells. It is not suitable to image samples disposed in the approximately 1-mm diameter wells of a 1536-well plate. Second, the geometric constraints of the angled illumination preclude the use of high numerical aperture collection optics, necessary to achieve sufficient sensitivity and resolution to image micron-sized objects, such as individual cells, at the bottom of the well.
Another approach to this problem uses a point-scan microscope. For example, in U.S. Pat. No. 5,547,849 to Baer et al., the use of a point-scan confocal system is taught. Baer et al. teach a method to increase the slow speed of image acquisition, inherent in point-scan confocal techniques, by sacrificing spatial resolution. If, for example, one expands the diameter of the illumination beam on the sample by a factor of 10, then the illumination area is increased 100-fold, permitting one to scan 100-times faster, under certain conditions. The speed increase is achieved, however, at the expense of resolution. Further, the detection devices appropriate to said scanning method, as disclosed in the '849 patent, are inferior, principally in terms of sensitivity, to those advantageously used in the present invention. Finally, the degree of background rejection is diminished along with the resolution. Thus the device disclosed in the '849 patent has lesser sensitivity, higher background and lower resolution than the present invention, all of which are important in the present application.
The present invention includes novel embodiments of a line-scan confocal microscope. Line-scan confocal microscopes are known in art. Two representative embodiments are the system disclosed by White et al. in U.S. Pat. No. 5,452,125 and that published by Brakenhoff and Visscher in J. Microscopy 171 17-26 (1993), shown in FIG. 7. Both use a scanning mirror to sweep the illumination across the sample. The same mirror de-scans the fluorescence radiation. After spatial filtering with a slit, the fluorescence is rescanned for viewing by eye. The use of the oscillating mirror enables these microscopes to rapidly scan a field-of-view. Line illumination is advantageous principally in applications requiring rapid imaging. The potential speed increase inherent in the parallelism of line illumination as compared to point illumination is, however, only realized if the imaging system is capable of detecting the light emitted from each point of the sample along the illumination line, simultaneously. An essential feature of the disclosed apparatus is the use of a detection device having manifold, independent detection elements in a plane conjugate to the object plane.
According to the present invention, the sample must lie in a “plane”, where the depth-of-field of the imaging system determines the precision of “planarity”. In a preferred embodiment, the imaged area is 1 mm2 and the depth-of-field is 10 μm. Thus, if the entire field is to be in focus simultaneously, the sample must be flat to 1 part in 100. This is true of many sample substrates (e.g. microtiter plates) over a local area (such as the central area of the well bottom). It is not practical, however, to require that the sample substrate be flat over its entire surface. For a microtiter plate having an extent of ˜100 mm, planarity of 1 part in 10,000 would be necessary.
The present invention provides for an optical autofocus system which maintains in “focus” the portion of the sample substrate being imaged. An optical autofocus mechanism has the advantage of being fast and being operational with non-conducting substrates such as plastic microtiter plates and microscope slides. Advantageously, this focus mechanism operates with negligible delay, that is, the response time of the focusing mechanism is short relative to the image acquisition-time, preferably a fraction of a second. Optically-based autofocus mechanisms suitable for the present application are known. For example, an astigmatic-lens-based system for the generation of a position error signal suitable for servo control is disclosed in Applied Optics 23 565-570 (1984), and a focus error detection system utilizing a “skew beam” is disclosed in SPIE 200 73-78 (1979). In a preferred embodiment of the present invention, the sample substrate is a microtiter plate. In this case, the preferred means of accomplishing the focusing depends further on the properties of the plate. If the thickness of the plate bottom were uniform to within a fraction of the depth-of-focus, then a focusing mechanism that maintained the plate bottom at a constant offset from the object plane would be adequate. Presently, commonly used microtiter plates are not sufficiently uniform. Thus, the focusing mechanism must track the surface on which the sample resides, which is typically the inside of the microtiter plate well. An aspect of the present invention is a novel autofocus mechanism for rapidly focusing on a discontinuous surface, such as the well bottom of a microtiter plate.
There is, therefore, a need for a method and apparatus for screening large numbers of chemical compounds accurately, quickly and inexpensively, in a homogeneous format. In addition, there is a need for a methods and apparatus that can perform multi-parameter fluorescence imaging with sufficient resolution to image individual cells and sub-cellular events. There is also a need for an imaging system that can additionally monitor a statistically significant population of cells at video-rates.